Abstract:

Recombinant human calpain-5 was expressed in insect cells using a baculovirus
system. The expressed calpain-5 was purified by both traditional chromatography and
by affinity-column chromatography. Both methods yielded active protease. Calpain-5
displayed very limited hydrophobicity. This indicated that calpain-5 is not a membrane
binding protein. Calpain-5 had pI of 8.3. The recombinant calpain-5 also exhibited
calcium-dependent proteolytic activity. The calculated calcium requirement for half-maximal
activity was 9.6 mM when incubated at 37°C and 26.5 mM when incubated at
30°C. Compared to traditional calpains, which require less than 1 mM calcium for half-maximal
activity, calpain-5 exhibited weaker proteolytic activity. This is an unusual
observation because calpain-5 lacks the typical calcium-binding domain of the calpains
and implied that other calcium-binding region of the protein account for calcium-binding
and sensitivity. Our results also showed that calpain-5 was different from
traditional calpains because its activity was higher at 37°C compared to 30°C and
remained active at 37°C for more than 2 hours. This differs from traditional calpains
which display better proteolytic activity at lower temperatures and become inactive
within 30 minutes of incubation in 37°C. Calpain-specific inhibitors, calpastatin and
E64, did not inhibit calpain-5. Only one calcium-binding inhibitor, PD150606, inhibited
calpain-5 proteolytic activity. These results confirmed that calpain's calcium-binding
domain is important in calpastatin binding and calpain-5 possesses other calcium-binding
regions. Calpain-5 was able to degrade spectrin, a ubiquitous cytoskeletal
protein. This indicates that calpain-5 might have a role in cell remodeling. Finally,
calpain-5 has the ability to degrade itself. It is not clear if this is the result of inter- or
intra-molecular proteolysis and whether this leads to activation of the protein or is,
instead, the first step in its degradation. Calpain-5 is expressed at highest concentrations
in testis, brain, liver and gastrointestinal tract. It is not clear why these tissues require a
unique calpain. Calpain-5 may provide these tissues with an additional calcium-dependent
proteolytic activity which is not regulated by calpastatin and which could
participate in cytoskeletal protein turnover.